CANDIDATE- I completed my graduate work at the University of Colorado, Boulder, focusing on endothelial cell dysfunction in human cohorts. After graduation, I broadened my research training by seeking a postdoctoral position in Dr. Victoria Bautch's (mentor) lab at the University of North Carolina (UNC), Department of Biology. Here, I use transgenic mouse and cell-based models to study the developmental and molecular mechanisms of vessel formation and dysfunction. At UNC, I am uniquely situated to carry out the proposed training plan and research strategy with the aid of my co-mentors, Dr.'s James Bear and Alexey Khodjakov. Completion of the proposed aims, training and educational activities will provide me with the necessary skills and collaborations to reach my long-term career goal of running an independent, extramurally funded biomedical research lab at a research-one university. PROPOSED RESEARCH- Setup and maintenance of blood vessels requires the integration and coordination of signaling pathways and cytoskeletal programs. Much is known about how aberrant signaling contributes to formation of pathological vasculature; however, less is understood about how cytoskeletal programs become dysfunctional and impair blood vessel architecture. This notion is best exemplified by tumor blood vessels; although, it is not limited to cancer-related pathologies. Tumor vessels are abnormal, leaky and dilated, providing a venue for tumor cell escape. Even in the absence of the tumor microenvironment, isolated tumor endothelial cells (ECs) demonstrate a preservation of abnormal cellular behaviors. These findings suggest that permanent alterations occur in tumor ECs, independent of signaling influences, possibly due to cytoskeletal abnormalities. In this regard, our group has previously described a mechanism by which excessive pro- angiogenic growth factor signaling, akin to that found in cancers, promotes the formation of supernumerary centrosomes (more than two centrosomes) in ECs. This data provided a mechanism for how tumor ECs acquire excess centrosomes in the tumor compartment at very high frequencies (>1/3 of total EC population). Furthering this finding, I have recently provided a novel mechanism linking interphase supernumerary centrosomes to EC motility defects in 2D (Kushner et al.; JCB. 2014). Our results demonstrated that supernumerary centrosomes are mispolarized, causing a cascade of cytoskeletal changes, which culminates in loss of directional cell migration. However, this investigation has prompted many additional questions, which this proposal strives to better understand and significantly expand upon. Globally, this proposal aims to determine how supernumerary centrosomes influence blood vessel morphogenesis in 3D sprouting (mentored phase). Furthermore, because centrosome polarization is vital for proper EC migration, I will also explore unique mechanisms of centrosome polarization and tethering (independent phase). For the mentored phase, in multiple models of 3D angiogenesis (in vitro, ex vivo, and in vivo) blood vessels with and without supernumerary centrosomes via Plk4 overexpression will be analyzed for morphological defects. Previously, I demonstrated that supernumerary centrosomes affect microtubule (MT) dynamics in 2D. To examine if MT defects persist in 3D, live-cell imaging and MT analysis software will be employed to monitor MT dynamics in ECs in 3D sprouts. Additionally, I hypothesize that supernumerary centrosomes will also effect the Golgi complex and vesicle trafficking, as these organelles are MT-dependent. Accordingly, the Golgi complex and vesicular proteins will be marked in ECs with fluorescent proteins in order to visualize their dynamics with and without excess centrosomes. If perturbed, key EC polarity and junctional proteins will be examined for mislocalization downstream of disrupted post-Golgi vesicle trafficking due to the presence of excess centrosomes. Predicted results will shed light on how supernumerary centrosome promotes blood vessel dysmorphogenesis in 3D. For the independent phase, I will characterize a unique phenomenon in which centrosome pairs (two centrosomes connected by MTs) can differentially regulate their MT dynamics in response to pulling forces exerted at the cortex, such as in cell migration. In this aim, I will explore how/if centrosomes sense tension using photoactivable Rac1 protein to induced membrane tension, software-based MT tracking and MT laser severing techniques. Candidate proteins involved will be selectively knocked down, overexpressed and rescued to thoroughly interrogate signaling programs responsible for modulation of centrosomal-MTs in response to tension cues. Lastly, a new mouse will be generated for conditional, vascular- specific knock down of dynein (a MT-motor protein) to explore how disruption of centrosome tethering and polarization impacts vessel network formation. .

Public Health Relevance

Oncologists target tumor blood vessels to hinder tumor progression by cutting of its nutrient supply; a medical approach called antiangiogenic therapy. Unfortunately, this method has not produced significant reductions in patient mortality, and often leads to the reoccurrence of more aggressive tumors. This finding underscores the need to better understand disease-related modification(s) to blood vessels. Disease-related changes in blood vessel biology may be unaccounted for when designing new therapies. This project aims to characterize one such disease modification, supernumerary centrosomes, a cytoskeletal defect that is present in very high frequencies in tumor endothelial cells. Results from this study may shed light on how tumor vessels develop and maintain abnormalities.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Career Transition Award (K99)
Project #
5K99HL124311-02
Application #
9140049
Study Section
Special Emphasis Panel (MTI (JA))
Program Officer
Wang, Wayne C
Project Start
2015-09-11
Project End
2017-08-31
Budget Start
2016-09-01
Budget End
2017-08-31
Support Year
2
Fiscal Year
2016
Total Cost
$119,136
Indirect Cost
$8,825
Name
University of North Carolina Chapel Hill
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Kushner, Erich J; Ferro, Luke S; Yu, Zhixian et al. (2016) Excess centrosomes perturb dynamic endothelial cell repolarization during blood vessel formation. Mol Biol Cell 27:1911-20